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Summary

One unique feature of vertebrate definitive hematopoiesis is the ontogenic
switching of hematopoietic stem cells from one anatomical compartment or niche
to another. In mice, hematopoietic stem cells are believed to originate in the
aorta-gonad-mesonephros (AGM), subsequently migrate to the fetal liver (FL)
and finally colonize the bone marrow (BM). Yet, the differentiation potential
of hematopoietic stem cells within early niches such as the AGM and FL remains
incompletely defined. Here, we present in vivo analysis to delineate the
differentiation potential of definitive hematopoietic stem/progenitor cells
(HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of
dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell
fate mapping and analysis of zebrafish runx1w84x and
vlad tepes (vltm651) mutants revealed that HSPCs
in the PBI gave rise to both erythroid and myeloid lineages. However, we
surprisingly found that HSPCs in the VDA were not quiescent but were uniquely
adapted to generate myeloid but not erythroid lineage cells. We further showed
that such distinct differentiation output of HSPCs was, at least in part,
ascribed to the different micro-environments present in these two niches. Our
results highlight the importance of niche in shaping the differentiation
output of developing HSPCs.

Within these distinct niches, hematopoietic stem cells appear to display
niche-specific differentiation repertoire
(Mikkola and Orkin, 2006;
Cumano and Godin, 2007). In BM,
hematopoietic stem cells undergo evident multilineage differentiation through
defined intermediates, with progressively restricted self-renewal and
differentiation capacity. Common myeloid progenitors and common lymphoid
progenitors arguably represent the earliest divergent point of hematopoietic
stem cell commitment from which erythromyeloid and lymphoid cells,
respectively, will be derived (Kondo et
al., 1997; Akashi et al.,
2000). Likewise, in the FL, hematopoietic stem cells proceed to
pronounced in situ differentiation into major blood lineages
(Cumano and Godin, 2007).
However, the differentiation hierarchy of hematopoietic stem cells in the FL
probably involves fetal intermediate progenitors that are distinct from those
in the BM, as common myeloid progenitors and common lymphoid progenitors
isolated from the FL exhibit less strict differentiation potential than do
those in BM (Mebius et al.,
2001; Traver et al.,
2001). In contrast to active multilineage differentiation
occurring in the FL and BM, AGM is generally regarded as a site that is devoid
of in situ differentiation (Cumano and
Godin, 2007). The most compelling evidence to support the
differentiation dormancy of hematopoietic stem cells in the AGM comes from in
vitro potential assays aimed to detect lineage-restricted progenitors
(Godin et al., 1999). These
assays fail to find the enrichment of intermediate precursors in the AGM and
most hematopoietic cells in this region are multipotent. However, the outcome
of such in vitro assays relies heavily on the applied culture condition, which
may not faithfully reflect a physiological context. Therefore, an in vivo
assay is needed to examine the differentiation capacity of hematopoietic stem
cells in the AGM.

By integrating the advantages for both embryological and genetic studies,
zebrafish provide unique opportunities to address early development-related
biological questions. Similar to higher vertebrates, zebrafish also experience
two successive waves of hematopoietic development: primitive and definitive
waves (Davidson and Zon, 2004;
de Jong and Zon, 2005).
Hematopoietic stem cells in zebrafish are thought to arise in the ventral wall
of dorsal aorta (VDA), as suggested by their expression of cmyb and
runx1 (Thompson et al.,
1998; Kalev-Zylinska et al.,
2002; Burns et al.,
2005; Gering and Patient,
2005) and by in vivo fate mapping
(Murayama et al., 2006;
Jin et al., 2007;
Kissa et al., 2008). We have
termed these presumed zebrafish definitive hematopoietic stem cells as
definitive hematopoietic stem/progenitor cells (HSPCs), acknowledging that
supporting functional data are still lacking. These VDA-originating HSPCs are
subsequently mobilized to an intermediate compartment, the posterior blood
island (PBI) or caudal hematopoietic tissue (CHT), prior to their final
colonization of the adult hematopoietic organ, the kidney
(Murayama et al., 2006;
Jin et al., 2007). Thus,
functional analogies of AGM and FL in zebrafish are very likely to be
represented by the VDA and PBI, respectively. However, to date, the
differentiation profile of HSPCs within the VDA and PBI is still poorly
defined.

Here, we presented an in vivo cell fate analysis in zebrafish embryos to
explore the lineage differentiation repertoire of HSPCs in the VDA and PBI,
with particular focus on their differentiation into erythroid and myeloid
lineages. We found that HSPCs were incapable of giving rise to erythroid
lineage in the VDA until they migrated to the PBI. However, to our surprise,
despite the inability of HSPCs to produce erythroid cells in the VDA, in situ
differentiation into myeloid lineages was readily detected in this region,
indicating that HSPCs in the AGM are not quiescent with respect to their
differentiation. We further showed that HSPCs lost the ability to give rise to
erythroid lineages when forced to remain in the VDA, suggesting that selective
emergence of definitive erythropoiesis in the PBI is at least partly due to
distinct micro-environment in the VDA and PBI.

MATERIALS AND METHODS

Maintenance of fish strains

Zebrafish were bred as described
(Westerfield, 1995). The
following fish strains were used: AB, Tg(fli1:eGFP)
(Lawson and Weinstein, 2002)
and vlad tepes (vltm651)
(Lyons et al., 2002). The
runx1W84X mutation was identified from F1 fish of
ENU-treated founders through genomic PCR and sequencing. The genomic
organization of zebrafish runx1 was determined using NCBI and Sanger
Center databases. Sequencing of exons of interest, data analysis and in vitro
fertilization to recover the mutation were performed as described previously
(Sood et al., 2006).

Morpholino injection

runx1 MO-1 (5′-TGTTAAACTCACGTCGTGGCTCTC-3′) and
runx1 MO-2 (5′-AATGTGTAAACTCACAGTGTAAAGC-3′) were
designed based on the sequence published by Burns et al.
(Burns et al., 2005). One-cell
stage embryos were injected with 2 nl morpholino solution at a concentration
of 0.6 mM runx1 MO-1 and 1 mM runx1 MO-2. The silent
heart MO was designed and injected according to a previous report
(Sehnert et al., 2002). A
standard control MO was obtained from Gene Tools.

May-Grunwald Giemsa staining

May-Grunwald Giemsa (Sigma) staining was performed as described previously
(Qian et al., 2007).

RESULTS

Definitive erythrocytes are enriched in the PBI from 3.5 dpf
onwards

To define the onset of definitive erythropoiesis, we first mapped the
temporal-spatial development of αe1-globin RNA-expressing cells
with whole-mount in situ hybridization. As αe1-globin RNA
expression was reported to be reactivated during later stages of development
(Brownlie et al., 2003),
detailed analysis of expression pattern of αe1-globin
transcript might aid in revealing the initiation of definitive erythropoiesis.
Consistent with a previous report (Brownlie
et al., 2003), we observed that the number ofα
e1-globin transcript-positive cells peaked at around 2 days
post-fertilization (dpf) and subsequently declined to a few cells by 3 dpf
(Fig. 1B,C). Such declination
from 2 dpf to 3 dpf reflects the cessation of αe1-globin
transcription in primitive erythrocytes. However, αe1-globin
RNA-expressing cells increased in number when embryos reached 3.5-4.0 dpf
(Fig. 1D). The propagation of
these cells appeared to be confined to the PBI, with their particular absence
in the VDA (Fig. 1D). By 5 dpf,
the αe1-globin RNA-expressing cells were also seen in the
kidney (Fig. 1E, arrow). This
late arising population of αe1-globin transcript-positive cells
probably represents newly generated cells of definitive erythroid lineage, as
their appearance temporally correlated with the emergence of circulating
erythroid precursors with a morphological characteristic similar to the
definitive proerythroblasts found in the adult kidney
(Traver et al., 2003) (see
Fig. S1 in the supplementary material). To illustrate convincingly the
definitive origin of these PBI restricted αe1-globin
RNA-positive cells, we probed αe1-globin transcript in embryos
in which definitive hematopoiesis was inhibited by either runx1
antisense morpholino oligonucleotide (MO) or a runx1 mutation in the
runx1w84x mutants. runx1w84x was
isolated by screening exons 3 and 4 of zebrafish runx1 gene through
genomic PCR and sequencing (Sood et al.,
2006). It harbors a G to A nucleotide substitution that converts a
Trp (amino acid 84) encoding triplet (UGG) to a premature stop codon (UGA)
(see Fig. S2A in the supplementary material). The resulting truncated protein
lacks majority of the Runt domain and therefore could be considered as null.
It is known that suppression of runx1 gene expression will
specifically abolish the formation of definitive HSPC and its derivatives
without affecting primitive hematopoiesis
(Okuda et al., 1996;
Burns et al., 2005;
Gering and Patient, 2005). In
line with this, we found that in the runx1 morphants and homozygous
runx1w84x mutants, cmyb-positive definitive
hematopoietic progenitors, including HSPCs, were absent from all definitive
hematopoietic tissues, including VDA, PBI and kidney (see Fig. S2B-E in the
supplementary material). Moreover, we observed that primitive erythroid cells
in the intermediate cell mass (ICM) were not perturbed in
runx1w84x mutants (compare Fig.
1F with
1A) and runx1 MO
knockdown embryos (morphants) (data not shown). By contrast,α
e1-globin RNA-positive cells in the PBI and circulating
definitive proerythroblasts were absent in runx1w84x
mutants (Fig. 1G) and
runx1 morphants (Fig.
1H) (n=46/52) (see Fig. S1C in the supplementary
material), showing that these cells are indeed of definitive origin.

Definitive erythropoiesis initiates from the PBI but not VDA

The enrichment of definitive erythroid cells in the PBI suggests that the
onset of definitive erythropoiesis in zebrafish occurs in the PBI rather than
the VDA. However, it could be conceived that abundant production of definitive
erythrocytes in the PBI is the consequence of the migration, proliferation and
differentiation of a small population of committed definitive erythroid
progenitors specified earlier in the VDA. This small cell population could be
masked by the presence of numerous primitive erythrocytes in wild-type
embryos. To clarify this possibility, we examined the expression profile ofα
e1-globin transcript in the vlad tepes
(vltm651) mutants, which harbor a nonsense mutation in the
essential erythroid regulator gata1
(Lyons et al., 2002). In
vltm651, primitive erythroid progenitors are specified
normally but subsequently depleted by 48 hpf, owing to accelerated apoptosis
(Lyons et al., 2002). As one
would anticipate that gata1 exerts similar functions during
definitive erythropoiesis, the vltm651 mutants could be
used to observe the emergence of the earliest committed definitive erythroid
progenitors, without the interference of primitive erythrocytes. As shown in
Fig. 1I,J, the number ofα
e1-globin RNA-positive cells in vltm651
embryos drastically reduced from a large number at 22 hpf to only a few cells
at 48 hpf. These rare cells in the 48 hpf vltm651 embryos
were randomly distributed, indicating that they were just vestige of primitive
erythrocytes. Hence, it is reasonable to believe that the reappearance ofα
e1-globin transcription after 48 hpf is a sign of the
committed definitive erythroid progenitors. As shown in
Fig. 1, albeit the magnitude is
low in vltm651 embryos compared with wild-type siblings,
the number of αe1-globin transcript-positive cells indeed
increased from 3 dpf onwards and reached its peak by 4 dpf
(Fig. 1K). More importantly,
these newly emerged definitive erythroid progenitors were first evident in the
PBI rather than VDA (Fig. 1K),
indicating that the commitment to definitive erythroid lineage took place in
the PBI. As expected, these PBI restricted αe1-globin
mRNA-positive cells in vltm651 significantly diminished
from 5 dpf onwards (data not shown), reflecting similar requirement of
gata1 during maturation of definitive erythroid lineage. Taken
together, the analysis of erythroid development in both wild-type and
vltm651embryos clearly support the argument that the
initiation of definitive erythropoiesis occurs in the PBI but not in the
VDA.

To determine whether PBI HSPCs that gave rise to definitive erythropoiesis
were originated from the VDA, we used a photo activatable cell tracer,
4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (flu), to label HSPCs in
the VDA, in order that their fates could be followed subsequently
(Vincent and O'Farrell, 1992;
Kozlowski et al., 1997;
Melby et al., 1996;
Serbedzija et al., 1998;
Keegan et al., 2004;
Jin et al., 2007). DMNB caged
flu was injected into one-cell stage Tg(fli1:eGFP) embryos in which
HSPCs were also marked by GFP (Jin et al.,
2007; Lawson and Weinstein,
2002). At 30 hpf, a small population (two or three) of
GFP-positive cells in the anterior part of VDA was uncaged with 405 nm laser
(Fig. 2A), and contribution of
these flu-labeled cells to definitive erythrocytes was examined by co-staining
of flu and αe1-globin RNA at 4 dpf
(Fig. 2B-F). We observed that
nine out of 28 uncaged embryos contained flu/αe1-globin
co-stained cells (Fig. 2C-F).
Remarkably, these flu/αe1-globin double-positive cells were
exclusively located in the PBI, confirming that HSPCs originated from the VDA
are capable of differentiating into erythroid cells only when they reach the
PBI region. Collectively, these data demonstrate that in the VDA HSPCs are
inactive with respect to their commitment and differentiation into definitive
erythroid lineage; this activity is later revived upon their homing to the
PBI.

VDA originated HSPCs are capable of giving rise to definitive erythroid
cells once homed to the PBI. (A) Lateral view of 30 hpf embryo
indicates the uncaged position (blue cross) in the anterior part of VDA.
(B) Lateral view of 4 dpf embryo. The boxed region (blue) indicates the
relative position in the PBI where flu and αe1-globin RNA
double-positive cells are found after uncaging. (C,D) Confocal
images of the boxed region in B show the flu signal andα
e1-globin RNA staining in the PBI. (E) Merged image of
C and D. (F) Superimposed view of E and DIC image. White arrows
indicate the co-staining of flu and αe1-globin RNA.

Definitive myeloid cells emerge in both the VDA and PBI

Because of the closely related ontogenic development of myeloid and
erythroid lineages during the adult phase of definitive hematopoiesis
(Akashi et al., 2000), we next
asked whether definitive myelopoiesis shared similar features to definitive
erythropoiesis during the early phase of zebrafish definitive hematopoiesis,
i.e. whether definitive myelopoiesis was also inactive in the VDA but became
activated in the PBI. To shed light on this, we examined the distribution of
myeloid cells using whole-mount in situ hybridization with differentiated
myeloid-specific markers, such as lyc, mpo and l-plastin
during zebrafish development (Herbomel et
al., 1999; Bennett et al.,
2001; Liu and Wen,
2002). An intriguing aspect of myeloid cell development was
revealed. At an earlier developmental stage, 22 hpf, myeloid cells were
restricted to the rostral part of the embryo, mainly scattered on the yolk sac
(data not shown). These cells are known to represent primitive myeloid cells
derived from the rostral blood island (RBI)
(Herbomel et al., 1999;
Lieschke et al., 2002). As
embryos developed, myeloid cells gradually emerged in the posterior part of
embryo with particular enrichment in the PBI and the region surrounding the
VDA (Fig. 3C,E,G; see Fig.
S3B,C,H,I in the supplementary material). The close proximity of myeloid cells
and HSPCs in the VDA raised an interesting possibility that these myeloid
cells were of definitive origin and derived from in situ differentiation of
HSPCs. To prove definitive hematopoietic origin of these myeloid cells, we
analyzed myeloid cell development in embryos with compromised definitive
hematopoiesis through repression of runx1 gene expression. Largely in
accordance with the dispensable role of runx1 in primitive
hematopoietic lineage development, we found that the expression of
l-plastin remained unaffected in runx1w84x mutant
(compare Fig. 3B with
3A) and runx1
morphants at 24 hpf (data not shown). Similarly, the expression of
lyc and mpo was not altered in runx1 morphants
(data not shown) but was slightly decreased in runx1w84x
mutants (see Fig. S3A,D,G,J in the supplementary material). Contrary to
largely intact primitive myeloid program in runx1-depleted embryos,
the number of myeloid cells in both the VDA and the PBI dramatically decreased
in 2 dpf runx1w84x mutants
(Fig. 3D) and runx1
morphants (data not shown, n=42/50), as well as 3 dpf
runx1w84x mutants (Fig.
3F; see Fig. S3E,K in the supplementary material). In fact,
virtually no myeloid cells were detected in VDA and PBI of the 5 dpf
runx1w84x mutants (Fig.
3H; see Fig. S3F,L in the supplementary material) and
runx1 morphants (data not shown, n=36/42). Thus, the
disappearance of myeloid cells in the VDA and PBI in the
runx1-suppressed embryos indicates that most if not all of this cell
population belong to definitive hematopoietic lineages.

Myeloid cells in the VDA are generated via in situ differentiation of
HSPCs

To confirm that HSPCs in the VDA could give rise to myeloid cells locally,
two or three GFP-positive cells in the anterior part of VDA of
Tg(fli1:eGFP) embryos were uncaged at 30 hpf and contribution of
these flu-labeled cells to myeloid lineage was examined by co-staining of flu
and L-plastin at 3 dpf. We observed that six out of 14 uncaged embryos
contained flu/L-plastin double-positive cells in the uncaged region (data not
shown), suggesting that the VDA was capable of generating myeloid cells. To
avoid inadvertently labeling myeloid cells originated from RBI and
unambiguously proving that these VDA-restricted myeloid cells were indeed
generated within the VDA region via in situ differentiation of HSPCs, rather
than seeded from other hematopoietic sites, we labeled HSPCs at 21 hpf, before
circulation started and before primitive myeloid cells migrated to the trunk
region (Herbomel et al., 1999;
Bennett et al., 2001;
Lieschke et al., 2002;
Liu and Wen, 2002). At 21 hpf,
HSPCs capable of giving rise to T cells are localized to intermediate cell
mass (ICM), a precursor to the VDA (Jin et
al., 2007). Two or three cells in the anterior ICM of 21 hpf
Tg(fli1:eGFP) embryos were uncaged
(Fig. 4A; see Fig. S4A in the
supplementary material), and differentiation of these labeled cells into
myeloid cells was determined with double staining against flu and L-plastin
protein or lyc RNA at 2 dpf or 3 dpf
(Fig. 4B-F′/G′; see
Fig. S4B-J in the supplementary material). In 24 uncaged embryos that survived
to 2 dpf, seven contained flu/L-plastin co-stained cells in the uncaged area
(four exclusively in the uncaged area and the other three in both uncaged and
PBI region) and one embryo contained flu/L-plastin double positive cells only
in the PBI region (Fig.
4B,D-D′/E′; Table
1). The average number of flu/L-plastin co-staining cells in each
of these embryos was 2-3. Likewise, when uncaged embryos were examined for
myeloid contribution at 3 dpf, nine out of 25 uncaged embryos had flu labeled
cells contributed to L-plastin+ myeloid cells in the original
uncaged region (five exclusively in the uncaged area and the other four in
both uncaged and PBI region) and two embryos were found to harbor
flu/L-plastin double-positive cells only in the PBI
(Fig. 4C,F-F′/G′;
Table 1). Similar results were
obtained by detecting co-localization of flu and lyc RNA (see Fig. S4
in the supplementary material; Table
1). Thus, as opposed to the apparent absence of definitive
erythropoiesis in the VDA, these in vivo tracing experiments demonstrate that
definitive myeloid cells do arise in the VDA via in situ differentiation of
HSPCs.

Definitive myeloid cells are generated in the VDA. (A)
Lateral view of 21 hpf embryo, indicating the uncaging position (blue cross)
in the anterior part of the ICM. (B,C) Lateral view of 2 dpf (B)
and 3 dpf (C) embryos. The boxed regions indicate the relative positions in
the VDA where flu and L-plastin protein double-positive cells were detected
after uncaging. (D,E) Confocal images of the boxed region in B
showing the flu signal and L-plastin staining in the VDA at 2 dpf after
uncaging at cross in A. (D/E) Merged image of D and E.
(D′/E′) Superimposed view of D/E and DIC image.
(F,G) Confocal images of the boxed region in C showing the flu
signal and L-plastin staining in the VDA at 3 dpf after uncaging at cross in
A. (F/G) Merged image of F and G. (F′/G′)
Superimposed view of F/G and DIC image. Staining for flu and L-plastin is
pseudopainted as green and red, respectively. Arrows indicate co-staining of
flu and L-plastin.

Extrinsic factors are essential in determining differentiation output
of HSPCs

The abovementioned lineage analysis clearly documents that HSPCs in the VDA
differ in their differentiation output from those in the PBI. Although HSPCs
give rise to only myeloid cells in the VDA, both erythroid and myeloid cells
are produced in the PBI. To explore the underlying basis for distinct lineage
output of HSPCs within different compartments, we therefore asked whether such
difference was solely controlled by a program intrinsic to HSPCs or whether
the micro-environment substantially contributed to this process. To shed light
on this, we scrutinized the expression of cmyb, a marker for early
definitive hematopoietic progenitors, including HSPCs, at 4 dpf, which is when
definitive erythrocytes have emerged in PBI. We noted that, in addition to the
PBI, presumptive HSPCs expressing cmyb but not the differentiation
markers l-plastin (Fig.
5A-I) and αe1-globin
(Fig. 1D,K) were present in the
VDA at 4 dpf. Unlike their counterparts in the PBI, these VDA-localized HSPCs
did not give rise to definitive erythroid lineages
(Fig. 1D,K). The co-existence
of definitive erythrocytes and HSPCs in the PBI but not in the VDA at 4 dpf
indicates that the PBI micro-environment may play a crucial role in initiating
definitive erythropoiesis.

To further substantiate the role of PBI in triggering definitive
erythropoiesis, we analyzed erythroid development in the sih (silent
heart) morphants, in which circulation was blocked by MO-mediated inhibition
of cardiac troponin T, thereby forcing HSPCs to remain in the VDA
(Sehnert et al., 2002;
Murayama et al., 2006). To
avoid interference from primitive erythropoiesis, sih MO was injected
into vltm651 embryos, in which primitive erythrocytes were
depleted without perturbation of early specification of definitive erythroid
progenitors. Consistent with previously reported experiments performed in
wild-type embryos (Murayama et al.,
2006), the initiation of HSPCs in the VDA was not affected in the
sih MO-injected vltm651 embryos, as detected by
runx1+/c-myb+ cells at 30 hpf
(Fig. 6A-D). HSPCs began to
accumulate in the VDA of sih MO-injected vltm651
embryos by 2 dpf (data not shown) and this accumulation became more evident at
3 dpf (Fig. 6F). In 4 dpf
sih MO-injected vltm651 embryos, these HSPCs
largely remained at the VDA, and were hardly detectable at the PBI
(Fig. 6H) (n=28/30).
Of note, the number of cmyb-positive HSPCs in 3 dpf and 4 dpf
sih MO-injected vltm651 embryos appeared
comparable with that in the controls (Fig.
6E-H), indicating normal progression of HSPC development. When
erythroid development was analyzed, αe1-globin mRNA-positive
definitive erythroid progenitors emerged normally in the PBI of 4 dpf control
MO injected vltm651 embryos
(Fig. 6I). However, in the
sih MO-injected vltm651 embryos, these definitive
erythroid progenitors were not detected either in the PBI or the VDA where
HSPCs were located (Fig. 6J)
(n=42/45). To demonstrate that the HSPCs trapped in the VDA were
still capable of definitive hematopoiesis, in situ hybridization for
lyc was performed in 4 dpf sih MO-injected
vltm651 embryos, in order to detect definitive myeloid
cells. As can be seen in Fig.
6K,L, lyc+ myeloid cells were readily detected, which
were preferentially localized to the VDA. Taken together, the data strongly
suggest that homing to PBI facilitates definitive erythropoiesis.
Intriguingly, some myeloid cells were still detectable in the PBI of
sih MO-injected vltm651 embryos. This could
result from circulation-independent migration of myeloid cells or from in situ
differentiation of committed erythroid/myeloid progenitors originated from the
caudal part of precirulation embryos
(Bertrand et al., 2007).

DISCUSSION

In the present study, we focused on the characterization of the in vivo
differentiation profile of HSPCs in the developing zebrafish embryos and
revealed that their differentiation into erythroid and myeloid lineages
differs substantially both in time and location. In situ differentiation of
HSPCs into definitive myeloid cells occurs as early as 2 dpf in VDA, the
zebrafish AGM analogy, whereas differentiation into erythroid lineages is
achieved only from 3-4 dpf onwards, when HSPCs have reached PBI, the zebrafish
FL analogy. Hence, our findings suggest that developing HSPCs elicit different
differentiation output as they home from one niche to another. During their
transition, HSPCs first undergo myeloid differentiation in the VDA and later
give rise to erythroid as well as myeloid lineages in the PBI. Recently, Kissa
et al. have suggested that cells in the VDA with low level of
cd41:eGFP expression are nascent zebrafish HSPCs
(Kissa et al., 2008).
Therefore, it would be of interest to track whether VDA localized definitive
myeloid cells and PBI-localized definitive erythrocytes are derivatives of
these cd41:eGFPlow cells, and, if so, to study the
differentiation behavior of these cells with respect to cell division pattern
as well as niche architecture.

We attribute selective emergence of definitive erythroid lineage in the
PBI, at least in part, to the different micro-environment between the VDA and
PBI, as HSPCs were unable to initiate definitive erythropoiesis when they were
trapped in the VDA owing to circulation defects in the sih morphants.
However, an ultimate demonstration for the impact of micro-environments on the
differentiation output of HSPCs would be performing reciprocal
transplantations with HSPCs isolated from these two sites. The success of such
transplantation demands stringent isolation of HSPCs to highly purified
fractions, which still awaits future technical advancement. At current stage,
the molecular basis for the role of environmental cues in triggering the onset
of definitive erythropoiesis in the PBI is not clear. It could be due to an
inhibitory effect imposed by the VDA or the presence of erythroid inductive
factors in the PBI. Although our data highlight the importance of
environmental cues in determining the onset of definitive erythropoiesis, we
could not underestimate the role of intrinsic program embedded in the
developing HSPCs, which cooperates with environmental factors.

Bertrand et al. (Bertrand et al.,
2007) recently reported the identification of committed
erythromyeloid progenitors in the caudal part of precirculation embryos
through which the first wave of definitive hematopoiesis initiates.
VDA-originated HSPCs and committed erythromyeloid progenitors were mapped to
anterior and posterior mesoderm, respectively, in the precirculation embryos,
suggesting they originate independently of each other
(Bertrand et al., 2007). In
addition, unlike the transient existence of committed erythromyeloid
progenitors, the number of which peaks at 30 hpf and declines from 40 hpf
onwards (Bertrand et al.,
2007), VDA-derived HSPCs probably represent self-renewing cells
that will eventually migrate to kidney where they sustain definitive
hematopoiesis throughout adulthood. Our study reveals that the differentiation
of VDA-originated HSPCs is distinct from that of committed erythromyeloid
progenitors. VDA-originated HSPCs elicit a niche-specific differentiation
repertoire: they generate myeloid cells via in situ differentiation in the VDA
but give rise to erythroid lineage later, after they home to the PBI. By
contrast, the differentiation of committed erythromyeloid progenitors is
always PBI restricted (Bertrand et al.,
2007). Hence, differentiation from VDA originated HSPCs provides
another source of myeloid and erythroid cells that is independent of those
generated through committed erythromyeloid progenitors born in the caudal
part. This additional wave of differentiation, which bridges between committed
erythromyeloid progenitors initiated definitive hematopoiesis and kidney
hematopoiesis, might reflect an evolutionary design to meet the need of
rapidly developing fish embryo.

The in situ generation of definitive myeloid cells in the zebrafish AGM
analogous region VDA is unexpected considering the currently held notion that,
in mice, the AGM is not a site for hematopoietic stem cell differentiation
(Godin et al., 1999;
Cumano and Godin, 2007).
However, our data are consistent with several published studies documenting
the presence or enrichment of myeloid restricted progenitors in the mouse or
chicken AGM (Cormier et al.,
1986; Cormier and
Dieter-Lievre, 1988; Ohmura et
al., 1999; Palis et al.,
1999). Among these, Palis et al.
(Palis et al., 1999) have
reported that compared with the rest of the embryo, AGM contained relatively
high proportion of myeloid progenitors such as Mac-CFC and Mast-CFC at 30- to
43-somite pairs and 60-somite pairs, respectively. By contrast, definitive
erythroid progenitors (BFU-E and CFU-E) do not display similar enrichment.
Thus, it appears that autonomous generation of definitive myeloid but not
erythroid cells in the AGM analogous region could be a common theme shared by
all vertebrates. It is still unclear whether other lineages besides myeloid
cells are produced in the AGM. The detection of ikaros expression, a
presumptive lymphoid progenitor marker, in the VDA might suggest the
co-existence of T cell progenitors in this region
(Willett et al., 2001).
However, further lineage tracing analysis is required to clarify this issue as
ikaros is also expressed in the multipotent progenitors
(Klug et al., 1998;
Georgopoulos, 2002).

The nature of VDA-derived definitive myeloid cells and their physiological
relevance remain to be elucidated. These myeloid cells may provide cytokines
that are essential for the survival and proliferation of the neighboring
HSPCs. A recent work by Robin et al. has revealed a crucial role for IL3 in
promoting the proliferation or survival of hematopoietic stem cells in the
AGM, although the identity of the IL3-producing cells was not determined in
their study (Robin et al.,
2006). Therefore, it is conceivable that myeloid cells derived
from hematopoietic stem cells in the AGM represent such nurturing cells
secreting paracrine growth factor for the stem cells. Alternatively, these
earlier arising definitive myeloid cells may consist of macrophage
populations, which are likely to be involved in promoting the maturation of
definitive erythroid cells later in the PBI, as mammalian macrophages are
reported to be indispensable for FL erythropoiesis
(Kawane et al., 2001). This
hypothesis is in accordance with our finding that VDA-derived myeloid cells
are already detectable in the PBI as early as 2 dpf prior to the appearance of
differentiated definitive erythrocytes in this site. Further investigations
are required to clarify these issues.

Supplementary material

Footnotes

We thank Jagman Chahal and Kevin Bishop for technical assistance. This work
was supported in part by the Intramural Research Program of
National Human Genome Research Institute,
NIH (P.P.L.) and by grant
from the Research Grants Council of Hong Kong
to Z.L.W. (662808). Deposited in PMC for release after
12 months.

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